DropFit: Determination of the Critical Concentration for Protein Liquid–Liquid Phase Separation

Journal of Molecular Biology Volume 437, Issue 19, 1 October 2025, 169294

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Determination of the critical concentration for protein liquid–liquid phase separation.

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Scale invariance of the droplet size distribution in the precritical region.

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The determination is made from accurate measurements away from the transition.

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Determination of the critical exponents to probe the universality class.

AbstractIn the liquid–liquid phase separation of proteins, dense liquid droplets often form within the dilute phase below the critical concentration. The resulting size distribution of these precritical droplets can be described by a scale-invariant distribution, which is characterized by an increasing average as the concentration approaches from below the critical value. This phenomenon can be leveraged for the quantitative estimation of the critical concentration. Here, to facilitate applications of this approach, we present the DropFit web server (https://www-cohsoftware.ch.cam.ac.uk/index.php/dropfit). DropFit can be used to estimate the critical concentration using experimental data on the length, area, or volume of the precritical droplets, which are taken away from the critical concentration and thus be more accurate and reproducible. We anticipate that the accurate value of the critical concentration under different experimental conditions will help understand the contributions from different macromolecules to the formation of protein condensates, and to investigate the perturbations that lead to pathological processes through the disruption of membraneless organelles.Graphical abstract